Light Sheet Fluorescence microscopy (LSFM) methods, such as Selective Plane Illumination Microscopy (SPIM), have had a big impact on the field of biological imaging since they provide fast, high-resolution optical sectioning over large tissue volumes with low photo-toxicity.
In fluorescence light sheet microscopy, the detection and illumination paths are orthogonal one to another. A sample is illuminated with visible light in a light-sheet volume around the focal plane of the detection optics so that fluorescence is generated only in a thin and well-defined “slice” of the sample.
Deschout H. et al. in “On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids”, published in Nanoscale, vol. 6(3), pages 1741-7 (2014), discloses a microfluidic chip with integrated waveguide for on-chip light sheet illumination for use in the technique fluorescence single particle tracking.
A review on fluorescence light sheet microscopy is given in “Selective plane illumination microscopy techniques in developmental biology” by Huisken, J. and Stainier, D. Y. R., published in Development 136 (12), pages 1963-1975 (2009).
In SPIM, the excitation light is focused by a cylindrical lens to a sheet of light that illuminates only the focal plane of the detection optics, so that no out-of-focus illumination is created (optical sectioning). For a single 2D image, no scanning of the sample is necessary. In SPIM, a stack of images is recorded by moving the sample through the light sheet, usually in a step-wise fashion. The samples, normally embedded in a gel, are imaged individually by mounting them one at a time on a sample holder of a microscope. SPIM was developed to generate multi-dimensional images of samples up to a few millimeters in size. In “Optical sectioning deep inside live embryos by selective plane illumination microscopy”, in Science 305, pages 1007-1009 (2004) by Huisken J. et al., the sample is embedded in a cylinder of agarose gel. The cylinder is immersed in an aqueous medium that fills the chamber, the excitation light enters the chamber through a thin glass window.
In US 2006/0033987, the object to be studied lies in the two-dimensional object illumination region when an image is recorded, the object being substantially larger than the thickness of this region. A two-dimensional image of the object parts located in this region is recorded by the two-dimensional detector. A three-dimensional image of the object is recorded by scanning the object in the detection direction through the stationary illumination region (or by scanning the illumination region through the object), a two-dimensional image being recorded in each position of the object.
In “A light sheet based high throughput 3D-imaging flow cytometer for phytoplankton analysis” by Wu J. et al., published in Optics Express, vol. 21(1:2), pages 14474-80 (2013), the authors report a light sheet fluorescence imaging flow cytometer for 31) sectioning of phytoplankton. The throughput of the instrument is quantified by the sample volume flow rate of 0.5 μl/min with a spatial resolution as achieved by light sheet microscopy.
Bruns T. et al. in “Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy”, published in Journal of Biomedical Optics, vol. 17(10) 2012, 101518, describes a device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. In the illumination device, the optical set-up for beam deflection and focusing, deflection mirror and cylindrical lens, is coupled to the objective turret of the microscope, whereas all other optical and mechanical components are fixed on the base plate of the microscope stage with a customized sample holder. According to the authors, the light sheet and the objective lens can be moved, simultaneously, in the vertical direction and all planes of the spheroid are imaged without re-adjustment of the microscope.
Applicant has observed that in the solution of Bruns T. et al. simultaneous movement of light sheet and objective lens without re-adjustment is possible provided that a pre-calibration of the capillary movement is performed.
A microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM) is described in “Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes” by Patra B. et al., published in Biomicrofluidics, vol. 8(5), 052109 (2014). The spheroid culture chambers are organized in a way such that only a single spheroid is illuminated at a time.
The capability of focusing light in microfluidic channels has attracted much interest in the recent years. This approach is known as optofluidics and describes the combination of optics and microfluidics. Furthermore, the tunability of optofluidic lenses makes them adaptive for a lab-on-a-chip system for biological and chemical analysis.
Paiè P. et al. in “Adaptable acylindrical microlenses fabricated by femtosecond laser micromachining”, Proc. SPIE 9355, Frontiers in Ultrafast Optics: Biomedical, Scientific, and Industrial Applications XV, 935516 (Mar. 9, 2015) describe an integrated microfluidic cylindrical in-plane lens with optical properties tunable by replacing the liquid used to fill the channel forming the lens. The design of the microlenses was optimised to reduce the effects of spherical aberrations in the focal region. The technique used to realize the device was femtosecond laser micromachining followed by chemical etching, which allows to easily fabricate 3D microfluidic devices with an arbitrary shape.
Osellame R. et al. in “Femtosecond laser microstructuring: an enabling tool for optofluidic lab-on-chips”, published in Laser & Photonics Reviews, vol. 5, pages 442-463 (2011), offer an overview on the technique of femtosecond laser micromachining.